Project Summary This project will advance a new mass spectrometry (MS) technology, charge detection mass spectrometry (CDMS) for analyzing large biomolecular assemblies in the 10 to 100 nm size range (i.e., having molecular weights of ~1 MDa to 1 GDa). Species in this size range, such as viruses and lipoproteins, play critical roles in human health. However, they are difficult to detect and characterize. Conventional MS instruments can determine masses and fragmentation patterns of many types of biomolecules, including assemblies; but, such measurements are limited to species below ~1 MDa. In CDMS, the masses of individual ions are directly determined from simultaneous measurement of each ion?s mass-to-charge ratio and charge. Proof-of-principle studies using our CDMS prototype, show that accurate masses can be determined into the MDa to GDa regime. As shown in this proposal, this is an enabling advance. However, our prototype design has a limited mass resolving power (the maximum we have measured is m/?m ~ 330). And, the time required for measuring a complete spectrum makes this instrument impractical for routine analyses. This project describes advances that will improve both the CDMS resolving power and spectrum acquisition speed ? each by at least an order of magnitude. This will allow high-resolution mass spectra for large species to be routinely recorded for the first time. We will also develop an ion mobility spectrometry (IMS) interface for CDMS. The IMS separation will improve the overall peak capacity and simplify the analysis of heterogeneous mixtures by CDMS analysis. In addition, the mobility of an ion depends on its structure and thus provides information that complements the charge and mass information from CDMS. The combined high-resolution IMS-CDMS instrument will provide a powerful new approach for the analysis of large biomolecules. Once calibrated and tested, the IMS-CDMS instrument will be used to investigate numerous problems important to human health. These include: virus assembly and disassembly, the characterization gene therapy vectors, drug and antibody binding to viruses, and the identification of lipoprotein subclasses. Such measurements have transformative potential. Viral gene therapy vectors, for example, are difficult to characterize because of their large sizes and because the genetic material is contained within virus capsids. Critical issues, such as whether capsids contain the full genome, partial genomes, or no genome, must be addressed. High-resolution IMS-CDMS measurements will provide information about small structural defects, which are difficult (if not impossible) to detect by other means. Similarly, IMS-CDMS analyses will impact other areas, such as cardiovascular diseases - the leading cause of death in the US. Plasma lipoproteins play a key role in the main underlying cause, atherosclerosis. However, the major classes, HDL, LDL, and VLDL, all exist as broad distributions of sizes and compositions. Delineation of subclasses will enable development of more reliable diagnostic tests and better therapeutics. Preliminary results indicate that high-resolution IMS-CDMS will be able to resolve key lipoprotein subclasses.